Compositions and methods for treating cancer

ABSTRACT

The invention provides compositions and methods to treat a hyperproliferative disorder with phenyl butyric acid (PBA) or a pharmaceutically acceptable salt thereof and an anti-cancer composition.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNos. 62/198,001 filed Jul. 28, 2015, and 62/241,379 filed Oct. 14, 2015,the entirety of which are incorporated herein by reference.

FEDERAL GRANT SUPPORT

The invention was made with government support under 1K25CA172218-01A1awarded by the National Institutes of Health and 2012-DN-130-NF0001awarded by the Department of Homeland Security. The government hascertain rights in the invention.

BACKGROUND

Melanoma is a cancer of the skin and is the fastest growing cancerincidence in the world today. Disease detected early can be removed bysurgery, but when melanoma spreads to other parts of the body (calledmetastatic melanoma) it is almost uniformly fatal. The reason for thisis that metastatic melanoma rapidly becomes resistance to all forms oftreatment. The first new therapy that appeared effective for melanomawas approved in 2011. The pharmaceutical called vemurafenib targetspatients with a gene mutation (BRAF^(V600E)) that is present in abouthalf of melanoma patients. Although these patients respond well to thetreatment, melanoma develops resistance to the therapy rapidly. Thus,the new therapy, which initially was heralded as the end of melanoma,extends life expectancy by only months with severe side effects.Vemurafenib is one of several BRAF inhibitors that are being used formelanoma therapy that target the BRAF protein. Melanoma developsresistance to all of these therapies. Several other drugs that havedifferent mechanisms of action are also approved for melanoma treatment,but the disease eventually develops resistance to all therapies formelanoma. There is no treatment for metastatic melanoma that overcomesresistance of melanoma cancer cells, which leads to a high mortalityrate.

Thus, there is a continuing need for compositions and methods for thetreatment of melanoma in animals (e.g., humans). Combination therapiesthat overcome resistance mechanisms that arise in almost all melanomapatients are particularly needed.

SUMMARY

In certain embodiments, the present invention provides a combination ofphenyl butyric acid (PBA) or a pharmaceutically acceptable salt thereof;and one or more anti-cancer compositions for the therapeutic treatmentof a hyperproliferative disorder. As a combined treatment thecombination treatment effectively destroys metastatic melanoma cancercells.

In certain embodiments, the hyperproliferative disorder is cancer. Incertain embodiments, the cancer is drug-resistant. As used herein, theterm “drug-resistant” is reduction in effectiveness of a drug in killingmalignant cells; reducing cancerous tumor size and rate of growth; andameliorating the disease or condition. In certain embodiments, thedrug's effectiveness is reduced by at least about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or even 100%, as compared to its effects whenfirst administered to the mammal.

In certain embodiments, the cancer is melanoma. In certain embodiments,the melanoma is resistant to vemurafenib treatment.

In certain embodiments, the PBA or a pharmaceutically acceptable saltthereof is administered simultaneously with the anti-cancer composition.

In certain embodiments, the PBA or a pharmaceutically acceptable saltthereof and the anti-cancer composition are administered sequentially.

In certain embodiments, the administration of the anti-cancercomposition begins about 1 to about 10 days before administration of thePBA or a pharmaceutically acceptable salt thereof.

In certain embodiments, the administration of the PBA or apharmaceutically acceptable salt thereof begins about 1 to about 10 daysbefore administration of the anti-cancer composition.

In certain embodiments, the administration of the PBA or apharmaceutically acceptable salt thereof and administration of theanti-cancer composition begins on the same day and/or simultaneously.

In certain embodiments, the anti-cancer composition comprisesvemurafenib.

In certain embodiments, the anti-cancer composition compriseschloroquine (or hydrochloroquin). In certain embodiments, theanti-cancer composition comprises a derivative of triphenylphosphonium(TPP), or a pharmaceutically acceptable salt thereof.

In certain embodiments, the anti-cancer composition comprises isipilimumab.

In certain embodiments, the PBA or a pharmaceutically acceptable saltthereof is administered in combination with vemurafenib, and the canceris melanoma.

In certain embodiments, the PBA or a pharmaceutically acceptable saltthereof is administered in combination with vemurafenib and chloroquine(or hydrochloroquin), and the cancer is melanoma.

In certain embodiments, the present invention provides a use of thecombination of PBA or a pharmaceutically acceptable salt thereof andanti-cancer composition in the preparation of a medicament for thetreatment of a hyperproliferative disorder in a mammal.

In certain embodiments, the present invention provides a kit comprisingPBA or a pharmaceutically acceptable salt thereof, a container, and apackage insert or label indicating the administration of the PBA or apharmaceutically acceptable salt thereof with an anti-cancer compositionfor treating a hyperproliferative disorder.

In certain embodiments, the present invention provides a productcomprising PBA or a pharmaceutically acceptable salt thereof, and ananti-cancer composition; as a combined preparation for separate,simultaneous or sequential use in the treatment of a hyperproliferativedisorder.

In certain embodiments, the present invention provides a method fortreating a hyperproliferative disorder in a mammal, comprisingadministering to the mammal a combination of PBA or a pharmaceuticallyacceptable salt thereof; and an anti-cancer composition.

In certain embodiments, the PBA or a pharmaceutically acceptable saltthereof is administered for more than a month.

In certain embodiments, the PBA or a pharmaceutically acceptable saltthereof is administered for more than a year.

In certain embodiments, the PBA or a pharmaceutically acceptable saltthereof is administered at a dosage of at least 500 mg/day.

In certain embodiments, the PBA or a pharmaceutically acceptable saltthereof is administered at a dosage of at least 1500 mg/day.

In certain embodiments, the PBA or a pharmaceutically acceptable saltthereof is administered via hose dose venous infusion to achieve aconstant blood concentration of at least 0.1 mM.

In certain embodiments, the present invention provides a use of PBA or apharmaceutically acceptable salt thereof; and an anti-cancer compositionfor the therapeutic treatment of a hyperproliferative disorder. Incertain embodiments, the hyperproliferative disorder is cancer. Incertain embodiments, the cancer is melanoma.

In certain embodiments, the PBA or a pharmaceutically acceptable saltthereof is administered in combination with an anti-cancer composition.

In certain embodiments, in the use:

a) PBA or a pharmaceutically acceptable salt thereof is administeredsimultaneously with the anti-cancer composition; or

b) PBA or a pharmaceutically acceptable salt thereof and the anti-cancercomposition are administered sequentially; or

c) administration of the an anti-cancer composition begins about 1 toabout 10 days before administration of the one or more anti-canceragents; or

d) administration of PBA or a pharmaceutically acceptable salt thereofbegins about 1 to about 10 days before administration of the anti-cancercomposition; or

e) administration of PBA or a pharmaceutically acceptable salt thereofand administration of the anti-cancer composition begins on the sameday.

In certain embodiments, the PBA or a pharmaceutically acceptable saltthereof is administered in combination with vemurafenib, and the canceris melanoma.

BRIEF DESCRIPTION OF THE DRAWINGS AND TABLES

FIG. 1: Proposed Mechanism of TPP-drug induced oxidative stress. Thepositively charged TPP head (A) preferentially translocates TPP to themitochondrial membrane due to the hyperpolarized membrane gradient incancer cell mitochondria; The side chain (B) (a 10 carbon aliphatic10-TPP shown here) embeds into the inner mitochondrial membrane,disrupting ETC complexes (drawn here at complex III) leading toincreased levels of superoxide and hydrogen peroxide. Structures of10-TPP and 12-TPP are also provided.

FIG. 2: Schematic representation of UPR. Under conditions of redoximbalance and nutrient stress, protein folding machinery is perturbed,which leads to the accumulation of mis-folded proteins in the ER andsubsequent ER stress. To manage ER stress, BiP (an ER resident molecularchaperone) initiates the UPR by activating IRE1, PERK and ATF6 mediatedsignaling pathways. These pathways reduce ER stress through increasedexpression of UPR related genes and molecular chaperones (ATF6 pathway);through activated ER associated protein degradation (ERAD) (IRE1pathway) to reduce ER protein load; and through reduced mRNA translationand autophagy in order to clear misfolded protein aggregates via theformation of autophagosomes (PERK pathway). If the ER stress cannot bereduced, UPR signaling pathways mediate the activation of apoptosis. AmyS. Lee, Cancer Res (2007); 77:3496-3499.

FIGS. 3A-3B: 10-TPP increases ROS production and cell-surface expressionof GRP78—an ER stress marker. a) Dihydroethidium (DHE) is a fluorescentmarker used to evaluate reactive oxygen species (ROS) production. Inthis experiment, we wanted to determine if 10-TPP increases cellular ROSlevels. A375 cells were treated with 1 μM 10-TPP for 24 h, 48 h, and 72h. Cells were then incubated with DHE and analyzed by flow cytometry.Results demonstrate that DHE oxidation increases with increased 10-TPPtreatment length, indicating increased production levels of ROS andsubsequent oxidative and ER stress. b) Glucose regulated protein GRP78is an ER resident protein. Under conditions of stress (ER stress,oxidative stress) it translocates to the cell surface. Uma K. Misra et.al., Journal of Biological Sciences (2002), Vol. 277, No. 44,42082-42089. We analyzed the cell surface expression of GRP78 as amarker of ER stress in A375 cells treated with 1 μM 10-TPP for 24 h, 48h, and 72 h. Following treatment, cells were harvested, labelled withAlexa-488 tagged GRP78 antibody, and analyzed by flow cytometry. Resultsdemonstrate that GRP 78 expression increases in cells treated with10-TPP and that expression increases with the length of 10-TPP treatmenttime. Collectively, these results demonstrate that 10-TPP increasesoxidative and ER stress, and that increased stress results in increasedGRP 78 cell-surface expression.

FIGS. 4A-4B: PBA and 10-TPP decreases ER stress levels and clonogenicsurvival in a time and concentration dependent manner. Malo A et al.,Pancreas (2013). March 42 (2):388. a) 4-phenyl butyric acid is known tobind with free nitrogen and therefore has the ability act as a chemicalmolecular chaperone. This experiment demonstrates the ability of PBA todecrease ER-stress. A375 cells were treated with PBA and 10-TPPrespectively for 48 hours. Our results demonstrate a decrease in theexpression of cell-surface GRP78 when treated with 0.5 μM and 1 μM10-TPP in combination with PBA. b) This result shows a decrease in theclonogenic survival of A375 melanoma cells when treated with thecombination of 10-TPP (0.5 μM, 1 μM) and PBA (100 μM, 500 μM and 1 mM)demonstrating the protective nature of ER stress

FIGS. 5A-5B: Combination treatment with 12-TPP and PBA decreases the ERstress and increases 12-TPP toxicity. Viability of cells was analyzedpost treatment of melanoma cells with 12-TPP and PBA (reduces ERstress). This result shows a decrease in the viability of SK-Mel-3melanoma cells when treated with 0.5 μM (FIG. 5A) and 0.25 μM (FIG. 5B)12-TPP alone along with combination concentrations of PBA (500 μM, 1 mM,2 mM, and 5 mM) for 6 h, 48 h and 72 h demonstrating the protectivenature of ER stress. In each experiment the treatments were as follows(from left to right): Control, 12-TPP, 12-TPP+500 μM PBA, 12-TPP+1 mMPBA, 12-TPP+2 mM PBA, or 12-TPP+5 mM PBA.

FIG. 6: PBA reduces ER stress.

FIG. 7: PBA combined with vemurafenib decreases the survival ofvemurafenib Sensitive A375 melanoma cells. In each experiment theconcentration in each treatment was as follows (from left to right):Control, 500 μM PBA, 1 mM PBA, 2 mM PBA, 5 mM PBA, Vemurafenib (5 μM),Vemurafenib+500 μM PBA, Vemurafenib+1 mM PBA, Vemurafenib+2 mM PBA, orVemurafenib+5 mM PBA.

FIG. 8: PBA combined with vemurafenib decreases the survival ofvemurafenib desistant A375 Melanoma cells. In each experiment theconcentration in each treatment was as follows (from left to right):Control, 500 μM PBA, 1 mM PBA, 2 mM PBA, 5 mM PBA.

FIG. 9: PBA combined with vemurafenib and chloroquine decreases thesurvival of vemurafenib resistant A375 Melanoma cells to a greaterextent than the combination of vemurafenib and PBA.

DETAILED DESCRIPTION

Melanoma is the dangerous type of skin cancer that develops in cellsthat produce melanin (melanocytes), usually presenting as an irregularspot/mole on the skin. Causes of melanoma include UV radiation and agenetic predisposition to this type of cancer. Unlike other cancers,prevalence of melanoma is increasing, with the highest occurrence amongindividuals 25-29 years old. The overall lifetime risk of developingmelanoma is 2.4%. In 2015, 73,870 new invasive melanomas are expected tobe diagnosed, with 9,940 people expected to die of melanoma. With earlytreatment, survival rate is 97%.

Melanoma can migrate to other parts of the body (metastatic melanoma),and one year survival rate drastically decreases with metastasis—15-20%for Stage IV. Current types of treatment include surgery, immunotherapy(Immune checkpoint inhibitors for advanced melanoma), chemotherapy,radiation therapy, targeted therapy (target cells with gene changes) andBRAF Inhibitors. BRAF is a protein kinase of the mitogen-activatedprotein kinase (MAPK) pathway, and it regulates cell growth,proliferation, and differentiation. Research suggests a BRAF^(V600E)mutation causes the BRAF protein (produced through the MAPK pathway) tobecome oncogenic. The mutation may lead to increased and uncontrolledcell proliferation, and resistance to apoptosis. The BRAF mutation isobserved in about 50% of melanoma tumors. Its presence is associatedwith poor prognosis in metastatic melanoma.

Melanoma is the fastest growing cancer incidence in the United States.Surgery is curative for melanoma confined to the skin, but metastaticmelanoma is lethal. Current FDA approved therapies for metastaticmelanoma (e.g., Vemurafenib, Ipilimumab), have increased life expectancyby months, however resistance develops rapidly. The exact mechanism bywhich drug resistance develops is unclear; however, autophagy is knownto play a major role. Autophagy is a self-degradative response of thecell towards nutrient stress. Conversely, autophagy also plays ahousekeeping role by removing mis-folded or aggregated proteins andclearing damaged organelles by forming autophagosomes. Thus, autophagyis believed to play an important role in tumor progression anddeveloping drug resistance during later stages of cancer. The UnfoldedProtein Response (UPR) mediated by GRP78 ER associated proteindegradation is one of the pathways that initiates autophagy in stressedcells. UPR involves the activation of three signaling pathways mediatedby IRE-1, PERK and ATF6. These pathways work towards decreasing theprotein load of ER by increasing the expression of molecular chaperons,activation of ERAD (ER associated protein degradation) and autophagy.However if the damage caused by the stress is extensive UPR signalingpathways initiate apoptosis. Amy S. Lee, Cancer Res (2007);77:3496-3499. Emerging evidence shows that in malignant cells ER stresscan be pro-survival and contribute to the development of drug resistanceby initiating autophagy.

A new combination therapy has been developed that kills vemurafenibresistant cells, but is virtually non-toxic to the rest of the body.

Phenylbutyrate, and Salts Thereof

As used herein, the term “Phenylbutyrate and salts thereof” includessalts of Phenylbutyrate. PBA has the following structure:

In certain embodiments, Phenylbutyrate is Buphenyl® (sodiumphenylbutyrate). Sodium phenylbutyrate is used for chronic management ofurea cycle disorders (UCDs). Its mechanism of action involves the quickmetabolization of sodium phenylbutyrate to phenylacetate. Phenylacetatethen conjugates with glutamine (via acetylation) to formphenylacetylglutamine, and phenylacetylglutamine is excreted by thekidneys. It has been observed that sodium phenylbutyrate reducesEndoplasmic Reticulum (ER) stress.

The cellular response to ER stress is neither fully oncogenic norcompletely tumor suppressive. It involves complex signaling with manypathways. The relative importance of each pathway varies between cellsdepending on chronicity of ER stress, and on relative expression ofvarious associated proteins. As solid cancers grow, nutrients and oxygenrequired exceed capacity of existing vascular bed, which can triggerangiogenesis (development of new blood vessels) to get moreoxygen/nutrients to the cancers. Cancers, however, usually becomehypoxic and nutrient-depleted, and with the hypoxia leading to impairedgeneration of ATP. The low ATP levels compromise ER protein foldingwhich leads to ER stress. Thus, unfolded, and/or misfolded proteins areassociated with ER stress and cancer cells exist with higher levels ofER stress relative to health cells.

Potential outcomes as a consequence of ER stress include high rates ofprotein synthesis that would trigger increased expression of autophagy,which is cytoprotective during stress (liberates amino acids, andremoves damaged organelles). Another outcome would be an increasedtolerance to hypoxia, which would promote tumor growth. This would alsoincrease autophagy, promoting drug resistance. Thus, a successfultreatment would inhibit autophagy and promote cell death.

Sodium phenylbutyrate decreases ER Stress. Lowering ER stress preventstolerance to hypoxia, and prevents cytoprotective autophagy (which leadsto drug resistance). Phenylbutyrate acts as a “chemical chaperone,”meaning it guides proper protein folding, and the presence of properlyfolded proteins lowers ER stress.

Anti-Cancer Agents

As used herein, the term “anti-cancer agent” includes therapeutic agentsthat kill cancer cells; slow tumor growth and cancer cell proliferation;and ameliorate or prevent one or more of the symptoms of cancer.

For example, the term “anti-cancer agent” includes vemurafenib andtriphenylphosphonates (TPP). Vemurafenib (Zelboraf®) is a cancer growthblocker and is a treatment for advanced melanoma.

Vemurafenib stops the proliferative effects of oncogenic BRAF protein.The standard method of administration is an oral tablet, administered 4×daily. Unfortunately, metastatic melanoma can resist vemurafenibtreatment. Vemurafenib slows tumor progression for only about 5.3months. As a result, finding an effective treatment for metastaticmelanoma is challenging.

For example, the term “anti-cancer agent” includes aTriphenylphosphonium (TPP) agent or derivative thereof that increasesreactive oxygen species (ROS) levels in cancer cell mitochondria, and apharmaceutically acceptable diluent or carrier. As used herein, the termtriphenylphosphonium (TPP) is any molecule containing atriphenylphosphine cation (⁺PPh₃) moiety. See, e.g., WO 2013/019975 andWO 2014/124384, which are incorporated by reference herein.

TPP salts can be reacted with alcohols, alkyl halides, and carboxylicacids, which allow them to be used as starting materials for thesynthesis of a large variety of chemical derivatives, e.g., XTPP agents.Charged molecules generally cannot pass through cell membranes withoutthe assistance of transporter proteins because of the large activationenergies need to remove of associated water molecules. In the TPPmolecules, however, the charge is distributed across the largelipophilic portion of the phosphonium ion, which significantly lowersthis energy requirement, and allows the TPP to pass through lipidmembranes. The phosphonium salts accumulate in mitochondria due to therelatively highly negative potential inside the mitochondrial matrix.The compositions of the present invention utilize XTPP agents that haveactivity in treating cancer cells, in that the XTPP agentspreferentially localize to cancer cells, as compared to the comparablenormal cells because cancer cells are often characterized by abnormalmitochondrial oxidative metabolism (Aykin-Burns N, Ahmad I M, Zhu Y,Oberley L W, and Spitz D R: Increased levels of superoxide and hydrogenperoxide mediate the differential susceptibility of cancer cells vs.normal cells to glucose deprivation. Biochem. J. 2009; 418:29-37. PMID:189376440) and altered mitochondrial membrane potential (Chen L B:Mitochondrial membrane potential in living cells, Ann. Rev. Cell Biol.1988; 4:155-81), relative to normal cells.

In certain embodiments, the TTP agent is 10-TTP or 12-TTP (see, FIG. 1).

In certain embodiments, the TTP agent is a compound of formula I:

Ph₃P⁺-L-WY⁻   I

wherein:

-   -   W is selected from:

L is absent, (C₁-C₁₂)alkyl, (C₁-C₁₂)alkylene, —(CH₂CH₂O)_(n)M-,—C(═O)NR^(L1)—, —NR^(L1)C(═O)— or —NR^(L1)C(═S)NR^(L1)—;

n is 1 to 12;

M is absent or —CH₂CH₂—;

R^(L1) is H or (C₁-C₆)alkyl;

R¹ is halo or —NHC(═O)R_(a);

R² is halo, SR_(b) or —C(═O)NHR_(c);

R³ is —NH(C═O)R_(d), —NH(C═O)NHR_(d) or phenyl wherein any phenyl of R³is optionally substituted with one or more halo, (C₁-C₃)alkyl,(C₁-C₃)haloalkyl, O(C₁-C₃)haloalkyl or —O(C₁-C₃)alkyl;

R⁴ is (C₁-C₆)alkyl or phenyl wherein any phenyl of R⁴ is optionallysubstituted with one or more halo, (C₁-C₃)alkyl, (C₁-C₃)haloalkyl or—O(C₁-C₃)alkyl;

R⁵ is —S(C₁-C₆)alkyl or —N((C₁-C₆)alkyl)₂;

R_(a) is phenyl optionally substituted with one or more halo,(C₁-C₃)alkyl, (C₁-C₃)haloalkyl or —O(C₁-C₃)alkyl;

R_(b) is phenyl optionally substituted with one or more halo,(C₁-C₃)alkyl, (C₁-C₃)haloalkyl or —O(C₁-C₃)alkyl;

R_(c) is phenyl optionally substituted with one or more halo,(C₁-C₃)alkyl, (C₁-C₃)haloalkyl or —O(C₁-C₃)alkyl;

R_(d) is phenyl optionally substituted with one or more halo,(C₁-C₃)alkyl, (C₁-C₃)haloalkyl or —O(C₁-C₃)alkyl; and

Y is a counterion;

or a pharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable diluent or carrier.

In certain embodiments, the anti-cancer agent is ipilimumab.

Compositions and Methods of Administration

The present invention provides a method for increasing the anticancereffects of a conventional cancer therapy (i.e., radio- and/orchemo-therapy) on cancerous cells in a mammal, comprising contacting thecancerous cell with an effective amount of PBA or a pharmaceuticallyacceptable salt thereof, and administering an additional conventionalcancer therapy modality. In certain embodiments, the additional cancertherapy is chemotherapy and/or radiation. In certain embodiments, thePBA or a pharmaceutically acceptable salt thereof and anti-cancer agentare administered sequentially to a mammal rather than in a singlecomposition. In certain embodiments, the mammal is a human.

In certain embodiments of the methods described above, the compositiondoes not significantly inhibit viability of comparable non-cancerouscells.

In certain embodiments of the methods described above, the cancer isbreast cancer, prostate cancer, lung cancer, pancreas cancer, head andneck cancer, ovarian cancer, brain cancer, colon cancer, hepatic cancer,skin cancer, leukemia, melanoma, endometrial cancer, neuroendocrinetumors, carcinoids, neuroblastoma, glioma, tumors arising from theneural crest, lymphoma, myeloma, or other malignancies characterized byaberrant mitochondrial hydroperoxide metabolism. In certain embodiments,the cancer is the above cancers that are not curable or not responsiveto other therapies. In certain embodiments the cancer is a melanoma. Incertain embodiments the cancer is a glioma.

In certain embodiments of the methods described above, the tumor isreduced in volume by at least 10%. In certain embodiments, the tumor isreduced by any amount between 1-100%. In certain embodiments, the tumoruptake of molecular imaging agents, such as fluorine-18 deoxyglucose,fluorine-18 thymidine or other suitable molecular imaging agent, isreduced by any amount between 1-100%. In certain embodiments the imagingagent is fluorine-18 deoxyglucose, fluorine-18 thymidine or othersuitable molecular imaging agent. In certain embodiments, the mammal'ssymptoms (such as flushing, nausea, fever, or other maladies associatedwith cancerous disease) are alleviated.

Administration of a compound as a pharmaceutically acceptable acid orbase salt may be appropriate. Examples of pharmaceutically acceptablesalts are organic acid addition salts formed with acids which form aphysiological acceptable anion, for example, tosylate, methanesulfonate,acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate,α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts mayalso be formed, including hydrochloride, sulfate, nitrate, bicarbonate,and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

Phenylbutyric acid (PBA) and the anti-cancer agents can be formulated aspharmaceutical compositions and administered to a mammalian host, suchas a human patient in a variety of forms adapted to the chosen route ofadministration, i.e., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes.

Thus, the present compounds may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet. For oral therapeutic administration, the active compound may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. Such compositions and preparations shouldcontain at least 0.1% of active compound. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60% of the weight of a givenunit dosage form. The amount of active compound in such therapeuticallyuseful compositions is such that an effective dosage level will beobtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, followed by filtersterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and the freeze drying techniques, which yield a powder ofthe active ingredient plus any additional desired ingredient present inthe previously sterile-filtered solutions.

For topical administration, the present compounds may be applied in pureform, i.e., when they are liquids. However, it may be desirable toadminister them to the skin as compositions or formulations, incombination with a dermatologically acceptable carrier, which may be asolid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the present compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the compounds of formula I to the skin are known to the art; forexample, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat.No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman(U.S. Pat. No. 4,820,508).

The dosage of the PBA or pharmaceutically acceptable salt thereof andthe anti-cancer agent will vary depending on age, weight, and conditionof the subject. Treatment may be initiated with small dosages containingless than optimal doses, and increased until a desired, or even anoptimal effect under the circumstances, is reached. In general, thedosage is about 450-600 mg/kg/day in patients weighing less than 20 kg,or 9.9-13.0 g/m²/day in larger patients. Higher or lower doses, however,are also contemplated and are, therefore, within the confines of thisinvention. A medical practitioner may prescribe a small dose and observethe effect on the subject's symptoms. Thereafter, he/she may increasethe dose if suitable. In general, the PBA or pharmaceutically acceptablesalt thereof and the anti-cancer agent are administered at aconcentration that will afford effective results without causing anyunduly harmful or deleterious side effects, and may be administeredeither as a single unit dose, or if desired in convenient subunitsadministered at suitable times.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. For example, thetherapeutic agent may be introduced directly into the cancer of interestvia direct injection. Additionally, examples of routes of administrationinclude oral, parenteral, e.g., intravenous, slow infusion, intradermal,subcutaneous, oral (e.g., ingestion or inhalation), transdermal(topical), transmucosal, and rectal administration. Such compositionstypically comprise the PBA or pharmaceutically acceptable salt thereofand the anti-cancer agent and a pharmaceutically acceptable carrier. Asused herein, “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, antibacterialand anti-fungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration, and a dietaryfood-based form. The use of such media and agents for pharmaceuticallyactive substances is well known in the art and food as a vehicle foradministration is well known in the art.

Solutions or suspensions can include the following components: a sterilediluent such as water for injection, saline solution (e.g., phosphatebuffered saline (PBS)), fixed oils, a polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),glycerine, or other synthetic solvents; antibacterial and antifungalagents such as parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol or sorbitol, and sodium chloride in the composition.Prolonged administration of the injectable compositions can be broughtabout by including an agent that delays absorption. Such agents include,for example, aluminum monostearate and gelatin. The parenteralpreparation can be enclosed in ampules, disposable syringes, or multipledose vials made of glass or plastic.

It may be advantageous to formulate compositions in dosage unit form forease of administration and uniformity of dosage. Dosage unit form asused herein refers to physically discrete units suited as unitarydosages for an individual to be treated; each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The dosage unit forms of the invention aredependent upon the amount of a compound necessary to produce the desiredeffect(s). The amount of a compound necessary can be formulated in asingle dose, or can be formulated in multiple dosage units. Treatmentmay require a one-time dose, or may require repeated doses.

“Systemic delivery,” as used herein, refers to delivery of an agent orcomposition that leads to a broad biodistribution of an active agentwithin an organism. Some techniques of administration can lead to thesystemic delivery of certain agents, but not others. Systemic deliverymeans that a useful, preferably therapeutic, amount of an agent isexposed to most parts of the body. To obtain broad biodistributiongenerally requires a blood lifetime such that the agent is not rapidlydegraded or cleared (such as by first pass organs (liver, lung, etc.) orby rapid, nonspecific cell binding) before reaching a disease sitedistal to the site of administration. Systemic delivery of lipidparticles can be by any means known in the art including, for example,intravenous, subcutaneous, and intraperitoneal. In a preferredembodiment, systemic delivery of lipid particles is by intravenousdelivery.

“Local delivery,” as used herein, refers to delivery of an active agentdirectly to a target site within an organism. For example, an agent canbe locally delivered by direct injection into a disease site, othertarget site, or a target organ such as the liver, heart, pancreas,kidney, and the like.

The term “mammal” refers to any mammalian species such as a human,mouse, rat, dog, cat, hamster, guinea pig, rabbit, livestock, and thelike.

The terms “treat” and “treatment” refer to both therapeutic treatmentand prophylactic or preventative measures, wherein the object is toprevent or decrease an undesired physiological change or disorder, suchas the development or spread of cancer. For purposes of this invention,beneficial or desired clinical results include, but are not limited to,alleviation of symptoms, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which thecondition or disorder is to be prevented.

The invention will now be illustrated by the following non-limitingExamples.

Example 1

Triphenylphosphonium (TPP) compounds with linear aliphatic side chainspreferentially accumulate in cancer cell mitochondria due to thehyperpolarized mitochondrial membrane potential, disturbing the cellularmetabolism leading to ROS (reactive oxygen species; e.g.superoxide/hydrogen peroxide) imbalance. This perturbs the redoxenvironment of endoplasmic reticulum (ER), consequently disturbingprotein folding machinery, which results in the accumulation ofmis-folded proteins and subsequent ER stress. In response to ER stressBiP (GRP78)—an ER resident molecular chaperone initiates an unregulatedprotein response (UPR) which works towards relieving the cell fromstress. This study aims to evaluate the protective nature of ER stressin melanoma and the toxicity of TPP compounds when combined with drugsthat decrease ER stress. Cell lines (A375 and SK-MEL-3) were treatedwith TPP derivatives in combination with 4-phenylbutyric acid (PBA),which acts as a chemical chaperone and reduces ER stress by decreasingthe protein load. Our results demonstrate reducing ER stress increasesthe cytotoxicity of TPP derivatives in metastatic melanoma cells,thereby demonstrating the protective nature of ER stress.

Emerging evidence suggest that ER stress could play an important role indrug resistance and disease progression. Evidence reveals that decreasedER stress levels, marked by number of viable cells, ROS productionlevels and Cell-Surface GRP78 expression, could increase thecytotoxicity of TPP derivatives in melanoma cells when treated incombination with other drugs (e.g. PBA) overtime. Combination therapiesare giving a better understanding of the protective nature of ER stress'and autophagy. A detailed understanding of the biochemical mechanism ofdrug resistance in melanoma can lead to a new paradigm in melanomatreatment that can improve the lives of a rapidly increasing number ofmetastatic melanoma patients.

Example 2

A potential combination therapy for Vemurafenib-resistant melanoma hasbeen developed. Vemurafenib is the standard of care for advanced stagemelanoma. Vemurafenib inhibits the action of BRAF^(V600E) therebyresulting in programmed cell death (apoptosis). However, resistancedevelops rapidly to treatment with Vemurafenib.

In certain embodiments, the invention involves the use of phenyl butyricacid or a pharmaceutical salt there of (e.g., a sodium salt) incombination with vemurafenib as a treatment for metastatic melanoma. Thediscovery is surprising because Buphenyl® is used for a completelydifferent disease treatment—the drug assists patients who havedifficulty removing nitrogen waste. Technically, sodium phenylbutyrateis a pro-drug and is rapidly metabolized to phenylacetate. Phenylacetateis a metabolically-active compound that conjugates with glutamine viaacetylation to form phenylacetylglutamine. Phenylacetylglutamine then isexcreted by the kidneys. On a molar basis, it is comparable to urea(each containing two moles of nitrogen). Therefore,phenylacetylglutamine provides an alternate vehicle for waste nitrogenexcretion.

In the present usage, Buphenyl® was found to reduce stress in theendoplasmic reticulum (ER). Cancer cells are generally under some stresslevels above normal cells, including oxidative stress, which in turncauses basal levels of ER stress. Interestingly, the initial response(within 24 hours) of introducing Buphenyl® to melanoma cells improvedtheir proliferation. However, somewhat fortuitously, it was discoveredthat when exposed to the drug for longer periods of time, vemurafenibresistant cells and cells sensitive to vemuafenib begin to die.According to the present cell survival assays, after about 6 days atlevels that are similar to the plasma levels reached by tabletadministration of Buphenyl®, these cells die. Further work was performedto understand the mechanism of this surprising finding and dataindicated that the mechanism involved suppression of a resistancemechanism called autophagy, which can help cells to be resistant to drugtreatments. Buphenyl® is normally given to patients in high doses fortheir entire life, which means that high dose Buphenyl® can beadministered to melanoma patients in combination withvemuarfenib—potentially without new side effects.

4-phenylbutyrate (PBA) reduces ER stress by acting as a chemicalmolecular chaperone. The sodium salt of PBA is an FDA approved drug(BUPHENYL®), which is administered to patients suffering from urea cycledisorder. The present inventors have shown that this drug has theability to kill vemurafenib-resistant melanoma cell lines, thus it canbe potentially administered in combination with vemurafenib for thetreatment of advanced stage melanoma. Studies showed that the levels ofCHOP (a marker of ER stress) decreases when A375 melanoma cells aretreated with a combination of vemurafenib and PBA (FIG. 6).

Experiments were also performed to determine the effect of PBA ontoxicity of Vemurafenib on A375 melanoma cell lines by measuringsurvival fraction through mean fluorescence intensity. It was observedthat as time increases the protective effect of vemurafenib decreases (6day, vemurafenib+PBA (5 mM)) (FIG. 7). It was also observed that boththe clonogenic survival and viability of vemurafenib resistant A375melanoma cells decrease with prolonged treatment with PBA (FIG. 8).

Example 3

Studies were performed to determine the clonogenic survival in an assayusing A375 cells. The cells were treated with vemurafenib, PBA and acombination of Vemurafenib, PBA and Chloroquine over a span of 6 days.1000 cells were re-played and incubated for 14 days days. Coloniesformed during this time were fixed using 70% ethanol and stained bycoumassi stain. The colonies were counted and normalized to un-treated(Control) plates. Vemurafenib and PBA both individually sensitizemelanoma cells. However, the combination with chloroquine issignificantly more effective that either of the treatments alone (FIG.9).

All publications, patents and patent applications cited herein areincorporated herein by reference. While in the foregoing specificationthis invention has been described in relation to certain embodimentsthereof, and many details have been set forth for purposes ofillustration, it will be apparent to those skilled in the art that theinvention is susceptible to additional embodiments and that certain ofthe details described herein may be varied considerably withoutdeparting from the basic principles of the invention.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The terms “comprising,” “having,”“including,” and “containing” are to be construed as open-ended terms(i.e., meaning “including, but not limited to”) unless otherwise noted.Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Embodiments of this invention are described herein. Variations of thoseembodiments may become apparent to those of ordinary skill in the artupon reading the foregoing description. The inventors expect skilledartisans to employ such variations as appropriate, and the inventorsintend for the invention to be practiced otherwise than as specificallydescribed herein. Accordingly, this invention includes all modificationsand equivalents of the subject matter recited in the claims appendedhereto as permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the invention unless otherwise indicated herein orotherwise clearly contradicted by context.

1. A combination of phenyl butyric acid (PBA) or a pharmaceuticallyacceptable salt thereof; and an anti-cancer composition comprising oneor more anti-cancer agents for the therapeutic treatment of ahyperproliferative disorder.
 2. The combination of claim 1, wherein thehyperproliferative disorder is cancer.
 3. The combination of claim 2,wherein the cancer is drug-resistant.
 4. The combination of claim 2,wherein the cancer is melanoma.
 5. The combination of claim 4, whereinthe melanoma is resistant to vemurafenib treatment.
 6. The combinationof claim 1, wherein the PBA or the pharmaceutically acceptable saltthereof is administered simultaneously with the anti-cancer composition.7. The combination claim 1, wherein the PBA or the pharmaceuticallyacceptable salt thereof and the anti-cancer composition are administeredsequentially.
 8. The combination of claim 1, wherein administration ofthe anti-cancer composition begins about 1 to about 10 days beforeadministration of the PBA or the pharmaceutically acceptable saltthereof.
 9. The combination of claim 1, wherein administration of thePBA or the pharmaceutically acceptable salt thereof begins about 1 toabout 10 days before administration of the anti-cancer composition. 10.The combination of claim 1, wherein administration of the PBA or thepharmaceutically acceptable salt thereof and administration of theanti-cancer composition begin on the same day.
 11. The combination ofclaim 1, wherein the anti-cancer composition comprises vemurafenib. 12.The combination of claim 1, wherein the anti-cancer compositioncomprises chloroquine or hydrochloroquin.
 13. The combination of claim1, wherein the anti-cancer composition comprises a derivative oftriphenylphosphonium (TPP).
 14. The combination of claim 1, wherein theanti-cancer composition comprises ipilimumab.
 15. The combination ofclaim 1, wherein PBA or the pharmaceutically acceptable salt thereof, isadministered in combination with vemurafenib, and the cancer ismelanoma.
 16. The combination of claim 1, wherein PBA or thepharmaceutically acceptable salt thereof, is administered in combinationwith vemurafenib and chloroquine or hydrochloroquin, and the cancer ismelanoma.
 17. (canceled)
 18. A kit comprising PBA or thepharmaceutically acceptable salt thereof, a container, and a packageinsert or label indicating the administration of the PBA or thepharmaceutically acceptable salt thereof with an anti-cancer compositionfor treating a hyperproliferative disorder.
 19. (canceled)
 20. A methodfor treating a hyperproliferative disorder in a mammal, comprisingadministering to the mammal a combination of PBA or the pharmaceuticallyacceptable salt thereof; and an anti-cancer composition.
 21. The methodof claim 20, wherein the PBA or pharmaceutically acceptable salt thereofis administered for more than a month.
 22. The method of claim 20,wherein the PBA or pharmaceutically acceptable salt thereof isadministered for more than a year.
 23. The method of claim 20, whereinthe PBA or pharmaceutically acceptable salt thereof is administered at adosage of at least 1500 mg/day. 24-30. (canceled)